In medical practice, identification of a disease requires not just recognition of the symptoms but also detecting specific features that would unambiguously indicate its presence. Furthermore, an early detection in asymptomatic populations is of utmost importance not only to facilitate early treatment but also to reduce health-care costs.
Usually, screening for signs of disease developments, biomarkers, is only conceivable through an analysis of biological fluids, such as blood, urine and cerebral spinal fluid, for circulating disease-related biomarkers. An accurate diagnostic can rarely be accomplished through the detection of just one single biomarker and a panel of markers has to be analyzed for a reliable results, such as in a multiplexed assay. Furthermore, monitoring the expression patterns of a variety of biomarkers at various stages of a disease could not only assist prognosis, but also allow one to follow disease progression.
Today, most protein biomarker assays are based on immunoassays. These usually provide a platform, made of either polymer or glass, bearing several immobilized antibodies spotted on different well-defined locations. These assays involve exposure of the platform to the sample followed by incubation with one or two further antibodies and several washing and blocking steps in between to increase the specificity of the assay results. Detection is usually via fluorescence detection, chromophoric absorption or a colorimetric readout. Importantly, conventional immunoassays (i.e. ELISA and fluorescent immunoassay) have limited expandability in terms of the number of proteins that can be detected per assay. This is attributed to the limited number of sensing area that can be incorporated within a single assay platform, due to the minimum laser spot-size achievable in the read-out system because of diffraction-limit, which impose a lower-limit to the useful size of a sensing area to a value not smaller than 200 nm, though in practice the size is usually in the range of 1 μm. Although one may argue that it is possible to modify a fluorescent immunoassay to allow multiple analytes (i.e. proteins/biomarkers) to be simultaneously detected by incorporating more than one fluorophore into each sensing area—for example, by expanding the number of protein-capturing fluorescent beacons used per sensing area, the broad fluorescence bandwidths (60-90 nm) unfortunately limit the maximum number of detectable fluorophores per sensing area to about 3. In other words, the maximum number of proteins detectable for each sensing area in a conventional immunoassay cannot exceed 3. Although, many immunosensor arrays have been developed in recent years, a truly rapid, accurate and miniaturizable system is still non-existing.
Vibrational spectroscopic techniques namely infra red (IR), normal Raman and Surface Enhanced Raman (SER) have been considered for analyte detection. Since near IR and mid IR technique suffers with the limitation of competing absorption from aqueous media, Raman spectroscopic techniques have evolved as the methods of choice. One important aspect of the Raman scattering is the correlation between the amount of the frequency shifts and the vibrational modes of the molecules. Since vibrational modes are sensitive to the chemical nature of the molecule, probing molecular vibrations can thus reveal information regarding its chemical geometry and interaction with other molecules. While a plethora of techniques, such as nuclear magnetic resonance (NMR) and X-ray crystallography, can also provide access to chemical structures, optical measurements of vibrational states via Raman scattering offer, owing to the ease of sample preparation, a much more convenient approach. For this reason, the Raman spectrum, which is unique to each molecule, has been utilized as a “fingerprint” in identifying unknown species, and in a more interesting aspect, Raman scattering is utilized for elucidating conformational changes.
However, under biological conditions the applications have been limited mainly due to the poor sensitivity and the need for high laser power and complicated instrumentation.
Most of these drawbacks were overcome by the development of Surface Enhanced Raman spectroscopy (SERS) where the spectral intensity is enhanced tremendously by the interaction of the SERS active analyte molecules with a substrate surface, e.g., a nanoparticle surface of copper, gold or silver. There are many cases where these enhancement factors are up to the level of single molecule detection (Nicholas & Ricardo, Chem. Soc. Rev., 2008, 37, 946-954). However, the detection of molecules with such extraordinary sensitivity still depends on the properties of the molecule-nanoparticle ensemble and is currently limited to certain classes of SERS active molecules.